This invention relates generally to the field of biochemistry, in particular to the isolation, cloning and expression of 1,3-dicarbonyl-dioxygenase.
Acetylacetone (synonyms: 2,4-Pentanedione, CAS No. 123-54-6; pentane-2,4-dione) is a widely used volatile industrial chemical. Studies of repeated exposure by peroral and inhalation routes have shown acute toxicity (Ballantyne B, Dodd D E, Myers R C, Nachreiner D J. 1986. Drug Chem Toxicol 9(2):133-46) including central neurotoxicity (Graham R H, Dodd D E. Ballantyne B. 2001. Vet Hum Toxicol 43(1):14-8) and possible immune system toxicity (Ballantyne B, et al. 1986, supra). Furthermore, fetotoxicity was observed (Tyl R W, Ballantyne B, Pritts I M, Garman R H, Fisher L C, France K A, McNeil D J. 1990. Toxicol Ind Health 6(3-4):461-74). The well characterized toxicology has made it a model compound for in vitro toxological investigations (Schmuck G, Schluter, G. 1996. Toxicol Ind Health 12(5):683-96).
On the other hand, diacylmethanes such as acetylacetone and derivatives have been shown to inhibit the mutagenic effect of various mutagenic substrates. As has been demonstrated in the model mechanism Salmonella typhimurium, acetylacetone, benzoylacetone and dibenzoylmethane inhibit the mutagenicity of 2-naphthohydroxamic acid; dibenzoylmethane and 1,3-indandione inhibit that of methylnitrosourea, benzo[a]pyrene and aflatoxin B1. The binding to tRNA of benzo[a]pyrene and aflatoxin B1 is inhibited by (a) benzoylacetone and dibenzoylmethane; and (b) dibenzoylmethane, 1,3-indandione and 1,1,1-trifluoroacetylacetone, respectively (Wang C Y, Lee M S, Zukowski K. 1991. Mutat Res 262(3):189-93). In the case of methylnitrosourea, a concomitant exposure to the inhibitors and the mutagen is necessary. These results demonstrate that active methylene compounds can inhibit mutagenicity and nucleic acid-binding of chemical carcinogens, presumably by trapping carcinogenic electrophiles, and can be potential anti-carcinogenic agents during the initiation stage.
Enzymes that cleave acetylacetone provide a means to reduce environmental contamination and acute toxicity of acetylacetone. Various enzymes acting on acetylacetone and other diketones have been previously described. One such enzyme from Pseudomonas sp. Strain VM15C hydrolyses acetylacetone to acetate and acetone (Sakai, K., Hamada, N., Watanabe, Y. 1985: Agric. Biol. Chem 49:1901-1902; and Sakai, K., Hamada, N., Watanabe, Y. 1986. Agric. Biol. Chem. 50:989-996). Although acetate can be easily utilized by the cell, acetone is not as easily introduced into metabolic pathways.
Several other enzymes, including acetylpyruvate hydrolase, catalyze the hydrolytic cleavage of beta-diketones. However, these enzymes are restricted to oxo-acid substrates and cannot cleave acetylacetone (Davey, J. F., Ribbons, D. W. 1975. J. Biol. Chem. 250:3826-3830; and Watson, G. K., Houghton, C., Cain, R. B. 1974. Biochem. J. 140:277-292).
Multi-step enzyme reactions not involving hydrolytic cleavage of acetylacetone are a possible means of acetylacetone degradation For example, a terminal oxidation step catalyzed by a cofactor-dependent monooxygenase, as is known for long-chain aliphatic hydrocarbon degradation (Gottschalk G., Bacterial Metabolism, Springer,1986), would result in a primary alcohol, which could be further oxidized to acetylpyruvic acid and integrated into the fatty acid degradation pathway. Also, subterminal oxidation, inserting an oxygen into acetylacetate by a monooxygenase in a Bayer-Villiger-like reaction, would give acetic-acid-2-oxo-ethyl-ester, which could be further hydrolyzed (Gottschalk G., 1986, supra; and Whyte L G, Hawari J, Zhou E, Bourbonniere L, Inniss W E, Greer C W. 1998. Appl Environ Microbiol 64(7):2578-84). However, cofactor-dependent multi-step enzyme reactions are relatively complicated detoxification systems.
For the foregoing reasons, there is a need for an enzyme capable of cleaving acetylacetone and related diketones to easily metabolized products in a single step.
The present invention provides a purified enzyme exhibiting 1,3-dicarbonyl-dioxygenase activity. In a preferred embodiment, the enzyme is purified from a strain of Acinetobacter johnsonii capable of growing on acetylacetone as the sole carbon source. In a most preferred embodiment, the isolated A. johnsonii enzyme is a multimeric protein of about 67 kilodaltons having subunits of about 16.6 kilodaltons.
The present invention provides an isolated polypeptide subunit of 1,3-dicarbonyl-dioxygenase. In a preferred embodiment, the subunit comprises the amino acid sequence set forth in SEQ ID NO: 2. Subunits comprising amino acid sequences having at least 70% sequence identity to SEQ ID NO: 2, determined by BLAST analysis, are included within the scope of the invention.
The present invention also provides an isolated polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2. A preferred embodiment comprises the nucleotide sequence set forth in SEQ ID NO: 1. Other embodiments include a polynucleotide encoding an amino acid sequence that is at least 70% identical to SEQ ID NO: 2, and a polynucleotide comprising a nucleotide sequence that is at least 60% identical to SEQ ID NO: 1. Sequence identity is determined by BLAST analysis.
The present invention further includes polynucleotides encoding 1,3-dicarbonyl-dioxygenase subunits in which the polynucleotide comprises one of the nucleotide sequences set forth in SEQ ID NOs: 3, 4, 8, 9, 10 and 11.
The present invention provides a vector containing the polynucleotide sequences described herein. Also provided are host cells containing a vector of this invention. Host cells can be mammalian cells, plant cells, insect cells, yeast and other fungi, or bacteria.
The present invention provides a process for producing a 1,3-dicarbonyl-dioxygenase. The process includes culturing a cell expressing the enzyme under appropriate conditions for producing the enzyme, and isolating the enzyme from the cell culture. The cell can be any cell expressing the enzyme. In a preferred embodiment, the cell is from a strain of A. johnsonii. In another preferred embodiment, the cell is a host cell containing a vector of this invention.
The present invention also provides a process for producing a subunit of a 1,3-dicarboryl-dioxygenase. The steps include culturing a cell expressing the subunit under appropriate conditions for producing the subunit, and isolating the subunit from the cell culture. The cell can be any cell expressing the subunit including a cell from a strain of A. johnsonii or a host cell containing a vector of this invention.
The present invention provides a composition containing a 1,3-dicarbonyl-dioxygenase. The enzyme can be mixed with buffers, salts, stabilizing agents, detergents and other components well known in the art to formulate products for the cleavage, decontamination or detoxification of acetylacetone and other 1,3-dicarbonyl compounds including cellular signaling substances based on homoserine lactones.
Finally, the present invention provides a method of cleaving a 1,3-dicarbonyl compound by reacting the compound with a 1,3-dicarbonyl-dioxygenase. The method can be used to decontaminate or detoxify 1,3-diketones, xcex2-ketoamides, xcex2-keto esters and 1,3-diesters.
The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.